DESCRIPTION (Abstract of the application) A major obstacle to successful stem cell therapy has been the low frequency of stem cell transduction with currently available factors. One strategy for overcoming this problem is to selectively expand transduced stem cells and their progeny in vivo. Hence, the overall objective of this project is to improve transduction efficiency in hematopoietic stem cells by developing strategies for selection of genetically marked stem cells in vivo. Most of the available selection markers such as DHFR and MDR1 require chemotherapeutic agents that act preferentially on dividing cells sparing the hematopoietic stem cell compartment, thereby limiting in vivo selection. Busulfan is a very potent stem cell toxin and is commonly used for eradication of host marrow in preparation for hematopoietic stem cell transplantation. Busulfan is remarkably stem cell selective in that bone marrow is eradicated well before other toxicities are evident. The action of busulfan is terminated by conjugation with glutathione S-transferases (GST). We have found that GST A1-1 is the most active busulfan conjugase of the human forms. We will therefore develop novel in vivo selection strategies using GST as a selectable marker and test whether in vivo selection of GST transduced hematopoietic stem cells can improve gene therapy for alpha-L-iduronidase deficient dogs as a model for stem cell gene therapy.